**1. Introduction**

Microorganisms are of primary importance in the agri-food industry. The knowledge of the microbial metabolic processes, as well as their behaviour and their technological characteristics, are required for any transformation process aiming to obtain healthy and quality foodstuffs. Wine production is also based on this assumption.

In oenology, the availability of yeasts able to drive alcoholic fermentation (AF) process and bacteria that efficiently carry out malolactic fermentation is required. In fact, in the first phase of the wine production process the yeasts, mostly belonging to the genus *Saccharomyces*, transform glucose into ethanol and carbon dioxide through the primary metabolism of sugars. Subsequently, lactic acid bacteria (LAB), usually *Oenococcus oeni* or *Lactobacillus* spp., metabolise malate into lactate, thus reducing the wine acidity [1, 2] and avoiding microbiological alteration.

In the past, fermentation of fruit juice, like those of apple and pear to produce cider, grape to obtain wine, or grains to make beer and so on for any kind of alcoholic beverages, have carried out by indigenous and naturally occurring microorganisms present in the original "must" [3–5].

The first molecular evidence in a Chinese Neolithic village, dated back to 7000 BC, shows that the food processing activity has given rise, without awareness, to the evolution of the genus *Saccharomyces* with the formation of new species, probably by interspecies hybridization or polyploidization [3]. Referring to *Saccharomyces cerevisiae*, its genetic evolution, which is due to human manufacturing, reflects the spread of grapevine cultivation and led to the origin of numerous strains [4–6].

Since the discovery of fermented beverages, their production process has undergone many evolutions, but initially the role of the microorganisms was unknown. Only in a second moment the choice of the best microorganisms to be used in a specific production, and their genetic improvement, become a conscious option. Hence, a certain degree of genetic yeast improvement was implemented in response to the requirements of wine production processes [3]. In fact, the scientific community proposed to the industry the use of starter cultures, that could be defined as a microbial (bacteria, yeast, mould) preparation containing a large number of live cells or resting forms of at least one species/strain that once added to a raw material leads to the production of a fermented food by accelerating and driving the fermentation process. The starter culture could contain unavoidable residues of additives and culture media [7–10].

Regarding wine production, until 150 years ago, also the transformation of grape must into wine took place without knowing the biological agent driving the fermentation process. In the usual cellar practices, it was carried out the inoculation of the must with a small amount of matrix from a previous successful fermentation, that in wine production was called "pied de cuve" [9]. In 1864, the role of microorganisms in fermentation was discovered by Louis Pasteur thus paving the way to the modern microbiology. Further research developments, achieved through microbiology, ecology, biochemistry and recently, molecular biology, have elucidated the metabolisms and in particular the biochemical process of alcoholic fermentation (**Figure 1**), as well as the interactions among microbial communities involved in winemaking, the phylogenetic and taxonomy. Based on this knowledge, the key role of yeasts in determining the quality of wine is now universally accepted [1, 11–13].

These scientific achievements have made it possible to supply oenological products and starter cultures appropriate for the industry. In fact, beginning from the mid-1960, the production and use of *S. cerevisiae* strains in form active dry yeasts (ADY) has expanded from California (United States) to the rest of the world [11–14]. In the major wine producing countries France, Italy, Spain, USA, Australia and Sud-Africa the use of ADY has almost fully replaced the spontaneous fermentation, especially in large-scale productions [3, 11, 13].

The importance of the adoption of yeast starter inoculation mainly consists in provide a faster beginning of AF. This is a stable and reproducible wine making procedure and, at the same time, ensures the absence of defects due to unwanted microorganism contamination [3, 9, 11]. The genetic selection of commercial ADY by the industry is based on the identification of specific technological and physiological features (**Table 1**) [3, 11, 15, 16].

The discovery of DNA, together with the development of molecular techniques further contributed to the taxonomic classification and, in a more practical context, to the identification of useful and spoilage microbes [17].

This also allowed the development of genetic improvement programs aiming at increasing genetic variability using diverse techniques (e.g. intra- or inter-specific

*An Overview on* Saccharomyces cerevisiae *Indigenous Strains Selection Methods DOI: http://dx.doi.org/10.5772/intechopen.99095*

#### **Figure 1.**

*Central metabolisms of alcoholic fermentation in yeasts.*


#### **Table 1.**

*General features to be considered in the selection of wine yeast.*

hybridization) and by genetic engineering techniques, mainly focused on improving the yeast qualitative characteristics [18–20]. In the last decades, genetically modified yeast was also obtained by insertion of useful genetic determinants of different species in *S. cerevisiae* genome [18, 21, 22].

More recently, a new technology to engineer the genome of microorganisms, based on CRISPR/Cas9 system, has been developed. Vigentini et al. [23] applied this editing system in engineering of wine yeast to obtain genotypes with low production of urea through the deletion of DNA coding for arginine permease.

This character is important because urea represent a precursor of ethyl-carbamate (EC) which is considered probably carcinogenic to humans [23–26].

Despite these scientific developments, the current appreciation of local, natural and organic food and wines by consumers has led again to the exploitation of spontaneous fermentation [27]. In fact, organic producers and some consumers consider the use of industrial yeast starter as a non-organic or non-natural practice. Moreover, due to the use of the same commercial strain for various wine style in different winemaking geographical areas, a standardisation of wine sensory characteristics is possible and negatively considered. These criticisms are justified, but, on the other hand, a spontaneous fermentation has to deal with the risks of loss quality related to potential stuck, uncontrolled microorganism development, spoilage and off-flavour production. These problems are only partially addressed by technological strategies aimed at controlling the process [8, 9]. Another aspect to be considered is the wine safety: the uncontrolled development of unwanted microorganisms could lead to the production of toxic compounds, such as biogenic amine, ethyl carbamate or mycotoxins which could negatively impact on human health [8, 9, 28].

As reported by the International Organisation of Vine and Wine (OIV), from winemaking point of view, there is a constant requirement to improve the wine style to answer to the consumer's demand for natural products and to compete in the globalised market [29–31]. As in the past, even today the scientific answers to these new market demands can be found by moving to specific yeasts selection. Massive propagations of yeast isolated from their own vineyard in order to inoculate the must, is an alternative strategy for winegrowers that combines unique sensory attributes with safe fermentations. Furthermore, the exploitation of indigenous yeasts is emerging as a marketing plan in several wine regions because the wines are perceived with more complex taste and flavour [9, 32].

The research of wild strains of *S. cerevisiae* to be applied in wine production processes started in the late 1990s. Other studies on non-*Saccharomyces* genus are currently performed in many regions of the world [33, 34]. The research of new strains is based on the need of new genotypes coming from genetic variability. As previously mentioned, different yeast strains can develop different secondary metabolites profile, therefore providing distinct character to the wine [32, 35].

A strategy to find *Saccharomyces* spp. genetic variability is to search it in the natural biodiversity of microflora present in the vineyard. Sampling in cellars would not be very fruitful for this purpose, because cellar premises and equipment could be heavily contaminated by commercial starters [36–38].

Based on these ideas, the approach of propagation of the autochthonous yeasts for wine production encounters the consumer needs as well as the main winemakers' target: terroir-yeast in the production of more complex tasting wines with a certain stylistic distinction, while preserving quality [36–38].

The aim of this chapter is to describe the methods applied for the selection of wine yeasts particularly on the indigenous *S. cerevisiae*. The possibility of using autochthonous yeasts is an innovative approach that increases the link with the terroir and a wine stylistic distinction. Moreover, it allows to obtain greater communication and product differentiation in terms of marketing.

### **2. Selection program of indigenous** *Saccharomyces cerevisiae* **strains**

Considering the oenological objectives described, the selection of indigenous yeasts must be planned and involves experiments aimed to isolate and propagate yeasts, and to test various oenological feature on laboratory and pilot scale (**Figure 2**).

*An Overview on* Saccharomyces cerevisiae *Indigenous Strains Selection Methods DOI: http://dx.doi.org/10.5772/intechopen.99095*

**Figure 2.** *Scheme of a selection process of indigenous* S. cerevisiae *yeasts.*

### **2.1 Yeast sampling in vineyard**

The vineyard soil would represent a reservoir of genetically different *Saccharomyces* spp. strains especially when the fruits are ripening and after the harvest. In fact, the increase of the number of fermentative yeasts during or near the harvest time has been recorded by molecular analysis, identification of culturable microorganisms and metagenomic approach [39, 40]. However, soil sampling at harvest time is not the optimal strategy for the isolation of wine yeast. The presence of *S. cerevisiae* in vineyard and at beginning of the fermentation process is sporadic [39–41]. In fact, yeasts belonging to the genus *Saccharomyces* spp. are not dominant on sound berries. The huge biodiversity of microflora living on bunch of grapes is related to insects and birds, that visit the ripe grapes [42]. *S. cerevisiae* strains are mainly detected during spontaneous fermentation when autochthonous grape yeasts and bacteria reduce their density due to the harsh environmental conditions represented by the high sugar content in must (realising a hypertonic living condition), and the increasing ethanol concentration in wine [32, 42]. To obtain an efficient selection of native yeasts, it is strongly recommended to start a spontaneous fermentation under controlled conditions [43, 44].

Several studies on spontaneous fermentations demonstrated the occurrence of an ecological succession with continuous shifts of the microbiota composition until the end of the process [42]. Due to the extreme condition of the must, especially high sugar concentration (250 g/l), low pH (3.5), nutrient availability and high osmotic pressure, the fermentative yeasts result to be more favoured compared to the species coming from the vineyard. *S. cerevisiae* is not dominant in this early step, but several fermentative yeasts such as *Metschnikowia pulcherrima*, *Hanseniaspora uvarum*, *Pichia* spp. and *Candida* spp. are detectable and carry on the alcoholic fermentation. The density of ethanol sensitive yeast species is reduced by the increase of alcohol concentration. *Zygosaccharomyces bailii*, *Torulaspora delbrueckii*, *C. stellata*, *C. zemplinina*, *Lachancea thermotolerans* can resist at 6–8% of ethanol, while *S. cerevisiae* proliferate vigorously up to consuming all the sugar and can easily tolerate up to 15–16% (V/V) of alcohol. After three days from AF start the *S. cerevisiae* population is in exponential growth phase (106 –107 colony forming units/ml). In the final step of alcoholic fermentation, over 10% of alcohol, the process is dominated by several *S. cerevisiae* strains. This stage is the most profitable to isolate the fermentative microflora and collect a certain number of genotypes belonging to *S. cerevisiae* species [35, 41].

Performing the grape harvest at ripening time allows to obtain a good degree of yeast biodiversity representing an excellent starting point for the strain selection [32, 43]. The practice of experimental scheme of grape sampling may vary according to the vineyard feature and economic considerations. In optimal situation, the criteria that could be respected have been described by Setati et al. [41]. In detail, it's recommended to:


As general principles, in the environment and in the vineyard agroecosystem too, yeast populations suffer from spatial and temporal fluctuation, so grape samples should be taken in several locations to gather a sufficient amount of *S. cerevisiae* strains that can be considered for the selection procedure [12, 37, 38]. It should be considered that damaged berries are a source of biodiversity for the sampling of fermentation yeasts [43].

Then, grape bunches should be placed in sterile bags avoiding the contamination with microorganisms unrelated to the sample, and transferred to the laboratory and processed as soon as possible according to the experimental protocol [41].

## **2.2** *S. cerevisiae* **strains isolation**

After the harvest of bunches, the spontaneous fermentation must be started, crushing the grapes. In order to avoid the contamination of the cultures, sterile conditions must be ensured by using sterilised or disposable equipment. In this step, di-ammonium phosphate (DAP) can be used as yeast nutrient and SO2 in the form of potassium metabisulphite can be added to promote the dominance of *S. cerevisiae* strain respect to SO2-sensitive non-*Saccharomyces*. Alternatively, the process could proceed without any addition of other nutrients or additive, except grape juice. The contact of must with berries skins is essential since the highest yeast concentration is in this compartment. Because of its resistance to osmotic pressure, tolerance to high sucrose concentration and to its efficient fermentation of sugar, *S. cerevisiae* is well adapted to the grape must [12, 42].

Due to the ethanol tolerance of *S. cerevisiae* and to the sensitivity of other yeast species, when the alcoholic fermentation is close to the end (ethanol more than 10% V/V), a sample of fermenting must-wine should be collected to isolate those yeasts that are driving the spontaneous process [12, 42]. Yeast isolation is performed by plating the collected samples on selective laboratory media in controlled conditions.

*An Overview on* Saccharomyces cerevisiae *Indigenous Strains Selection Methods DOI: http://dx.doi.org/10.5772/intechopen.99095*

The dilution of fermenting must or wine at the end of AF is critical to evaluate a reasonable number of colonies in the solid artificial media. However, a compromise with the risk to lose biodiversity with the dilution procedure must be found, so that the sample should represent the yeast population in each vinification. Usually, the sample is diluted until 10−5 or 10−6 and aliquots of these suspensions are plated. Wallestein Laboratory (WL) agar solid media allowing to differentiate among yeast species on the basis of different colours of the colonies is usually used for yeast growth (**Figure 3**). The incubation temperature must be 24–26° C.

The genotypes loss during the isolation phase, is a problem to deal with during the selection procedure. As the different *S. cerevisiae* strains are morphologically indistinguishable, the colonies must be sampled randomly in plates with 250 colonies maximum. A total of 24–30 colonies for each plate must be sampled and analysed by molecular techniques for species assignation and strain differentiation [46]. Once the isolation and genetic identification phases have been completed, the strains are usually long term stored at −80° C in glycerol 50% V/V to preserve membrane integrity [32, 41, 47] and in slant with YEPD (Yeast Extract Peptone Dextrose) solid agar for short term conservation at 4°C. This procedure has been applied in several studies such as Capece et al. [43], Efstratios et al. [48], Viel et al. [49].

## **2.3 Genotyping: Molecular biology applied to yeast species identification and**  *S. cerevisiae* **strain characterisation**

One of the main goals in microbiology is to obtain a valid identification of microorganisms. Traditionally, before the application of molecular biology techniques, yeasts have been identified by morphological and physiological criteria. These methods are basically labor-intensive, time-consuming, and usually provide doubtful identifications. This is due to similar colony morphology, to the influence of culture conditions on yeast physiology and to the presence of different teleomorphic and anamorphic forms in the same species [50, 51].

The progress in molecular biology allowed to develop fast and efficient methods to identify both species and strains. Methods based on DNA technique,

**Figure 3.** *Some* S. cerevisiae *colonies on Wallestein laboratory (WL) agar medium.*

some of these based on DNA Polymerase Chain Reaction (PCR) proved to be the most effective identification tool. Allozyme patterns, DNA–DNA hybridization, electrophoretic karyotyping, microsatellite analysis, nested-PCR, random amplified polymorphic DNA (RAPD) and mitochondrial DNA restriction analysis are the molecular biology techniques which first contributed to yeast identification [50–58]. As an example, electrophoretic karyotyping is based on the weight analysis of the yeast entire genome according to the species [52]. Other examples of molecular analysis are: insertion site polymorphism of delta elements, simple nucleotide polymorphism (SNP), amplified fragment length polymorphism (AFLP), intron splice sequence amplification, PCR of intron of mitochondrial genes, ribosomal DNA sequencing [12, 54, 57, 59, 60].

Moreover, the genome of *S. cerevisiae* S288C, a model organism in both cell biology and medicine, was entirely sequenced in 1996 and this reference DNA is at the base of the *Saccharomyces* Genome Database (SGD). This achievement facilitates the introduction of new molecular techniques [61, 62].

In this paragraph we will describe more in detail the most relevant techniques for the identification and characterisation of *S. cerevisiae*. RAPD is a PCR based technology in which DNA polymorphism is analysed by amplifying random DNA segments with single primers with an arbitrary nucleotide sequence. A single primer is used to anneal to the genomic DNA at different sites.

Quesada and Cenis in 1995 [53] and Baleiras Couto et al. in 1996 [54] used this method in the taxonomic identification of wine yeast strains both at genera and species level [53, 54]. In 2010, Capece et al. have used a RAPD-PCR with M13 primer to execute a fingerprint on 341 isolates obtaining 130 indigenous strains [43]. This technique can be applied both for interspecific and intraspecific characterisation [55]. The advantage of using RAPD is that it is rapid and easy to assay and there is no need of knowing the DNA sequence, but the main drawback is the low reproducibility.

In 1994, some authors focused the attention on mitochondrial DNA (mtDNA) for fast characterisation of *Saccharomyces sensu stricto complex* [49, 63]. The high polymorphism of this DNA can be highlighted after restriction enzymes digestion (endonucleases: *AluI, DdeI, HinfI, RsaI*). The resulting mtDNA band patterns is species-specific and allows the identification of *S. cerevisiae*, *S. bayanus*, *S. paradoxus*, *S. pastorianus* species [63]. The mtDNA restriction analysis (RFLP-mtDNA) was also applied in many experimentations at strain level due to high degree of intraspecific heterogeneity [42, 47, 64].

For the identification at species level, the main used technique is based on the amplification of the rDNA Internal Transcribe Spacer (ITS) region and subsequent digestion with restriction enzymes. This is a specific type of RFLP also called Amplified Ribosomal DNA Restriction Analysis (ARDRA). The amplified target region includes the conserved gene coding for the 5.8 rRNA subunit and the two flanking non-coding and variable internal transcribed spacers named ITS1 and ITS2 [64, 65].

This method was described by Guillamón et al. in 1998 [64], Granchi et al. [50] and Esteve-Zarzoso et al. in 1999 [51] and is used in oenological yeast species identification still today [50, 51, 64, 65]. According to Guillamón et al. [64], the method is based on a first step of amplification targeting the nuclear rRNA gene region by using primers ITS1 and ITS4. This region includes the coding zone for the RNA ribosomal 5.8S and two non-coding regions at its ends (ITS1 and ITS2) (**Figure 4**). PCR products show a high length variation according to the different species leading to a preliminary discrimination among yeasts after agarose gel electrophoresis. The second step consists in PCR product digestion using three enzymes, endonucleases, *Hinf*I, *Cfo*I and *Hae*III. Each species shows a specific restriction pattern

*An Overview on* Saccharomyces cerevisiae *Indigenous Strains Selection Methods DOI: http://dx.doi.org/10.5772/intechopen.99095*

#### **Figure 4.**

*Nuclear rRNA gene and region of DNA amplification through PCR using primer ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATA TGC-3′).*

according to each endonuclease. So that a discrimination at species level is easily obtained. Thanks to this method it was possible to distinguish with confidence the presence for example of *Hanseniaspora uvarum*, *Candida stellata*, *C. vini*, *S. cerevisiae*, *S. paradoxus*, *S. bayanus*, etc. during spontaneous must fermentation [51, 64, 65]. Similar results have been obtained by Esteve-Zarzoso et al. [51] who analysed 243 different strains belonging to 132 different species, from the Spanish Type Culture Collection (CECT). In the experiment the amplicon digestion has carried out using *HinfI*, *CfoI* and *HaeIII* and other four endonucleases (*AluI*, *TaqI*, *DdeI* and *ScrFI*). This second set of endonouclease was necessary in some particular cases where more restriction patterns were required to get an efficient identification.

In general, this technique is highly reproducible and allows the discrimination of large number of samples.

Focusing on *S. cerevisiae* strain discrimination, inter-delta analysis and microsatellite polymorphism analysis represent useful and easy-to-use molecular tools. Inter-delta regions are some repetitive DNA sequences in *S. cerevisiae* genome, often associated with the transposon Ty1. These regions can be used for the genetic identification of *S. cerevisiae* strains thanks to their different number and location within the species by amplifying these regions with specific primers. Several authors studied inter-delta fingerprinting of *S. cerevisiae* strains and showed that PCR-amplification of DNA delta sequences is a reproducible, strain-specific and simple method that can be successfully applied to monitor strain population dynamics in wine fermentation [47, 66–68].

Microsatellite markers, based on Simple Sequence Repeats (SSRs) scattered throughout the genome [69–73], represent the "gold standard" for this discrimination. Microsatellites are short DNA motifs, 2–6 bases (e.g GATA, GACA, etc.), tandemly repeated five to fifty times (**Table 2**). Their sequence lengths are intraand interspecific polymorphic across species [56, 69–73]. Moreover, SSRs are characterised by higher mutation rate than the rest of the genome, representing a formidable tool for the genetic differentiation of *S. cerevisiae* strains, as reported by



*Some simple sequence repeat motif and primers' origin and sequence for* Saccharomyces cerevisiae *typing.*
